Quantitation of antigen in tissue by immunofluorescence image analysis

John M. Basgen; Thomas E Nevins; Alfred F. Michael

doi:10.1016/0022-1759(89)90188-9

Quantitation of antigen in tissue by immunofluorescence image analysis

John M. Basgen, Thomas E Nevins, Alfred F. Michael

Pediatric Nephrology

Research output: Contribution to journal › Article › peer-review

13 Scopus citations

Abstract

A method is described to measure antigen(s) in tissue section using an image analysis system to quantitate immunofluorescence following staining with fluorescein isothiocyanate (FITC)-conjugated antibody. The antigen utilized was an ¹²⁵I-labeled goat anti-glomerular basement membrane antibody (1409 cpm/μg) administered intravenously to each of 11 Sprague-Dawley rats in doses ranging from 1.09 to 34.72 mg IgG. 24 h later, both kidneys were obtained for quantitative immunofluorescence following staining of tissue sections with FITC-labeled rabbit anti-goat IgG and for determination of radioactivity which reflects the amount of goat IgG present in isolated glomeruli. A linear correlation (r = 0.97) was observed between the dose of administered goat ¹²⁵I-IgG and the amount bound to isolated glomeruli over the entire dosage range. A highly significant correlation (r = 0.98) was also seen between the mean brightness per glomerulus as determined by quantitative immunofluorescence and the amount of ¹²⁵I-IgG bound per glomerulus but only at values less than 500 pg of IgG per glomerulus. Above these levels no correlation was observed, suggesting the presence of hidden epitopes in the bound goat IgG or the lack of availability of the FITC-labeled rabbit antibody.

Original language	English (US)
Pages (from-to)	77-83
Number of pages	7
Journal	Journal of Immunological Methods
Volume	124
Issue number	1
DOIs	https://doi.org/10.1016/0022-1759(89)90188-9
State	Published - Nov 13 1989

Bibliographical note

Funding Information:
We thank Christine Alonzi, Tom Groppoli, Sue Kupcho-Sandburg and Karen Clugge for excellent technical assistance, Paul Walker for the preparation and quantitation of radiolabeled IgG. We are also grateful to the staff of the Biomedical Image Processing Laboratory for help and assistance with the image analysis system and Dr. Ruth 0sterby for critical reading of the manuscript. This work was supported by grants from the National Institutes of Health (Al10704) and the Viking Children's Fund.

Keywords

(Kidney)
Basement membrane
Fluorescence
Image processing
Immunohistochemistry

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title = "Quantitation of antigen in tissue by immunofluorescence image analysis",

abstract = "A method is described to measure antigen(s) in tissue section using an image analysis system to quantitate immunofluorescence following staining with fluorescein isothiocyanate (FITC)-conjugated antibody. The antigen utilized was an 125I-labeled goat anti-glomerular basement membrane antibody (1409 cpm/μg) administered intravenously to each of 11 Sprague-Dawley rats in doses ranging from 1.09 to 34.72 mg IgG. 24 h later, both kidneys were obtained for quantitative immunofluorescence following staining of tissue sections with FITC-labeled rabbit anti-goat IgG and for determination of radioactivity which reflects the amount of goat IgG present in isolated glomeruli. A linear correlation (r = 0.97) was observed between the dose of administered goat 125I-IgG and the amount bound to isolated glomeruli over the entire dosage range. A highly significant correlation (r = 0.98) was also seen between the mean brightness per glomerulus as determined by quantitative immunofluorescence and the amount of 125I-IgG bound per glomerulus but only at values less than 500 pg of IgG per glomerulus. Above these levels no correlation was observed, suggesting the presence of hidden epitopes in the bound goat IgG or the lack of availability of the FITC-labeled rabbit antibody.",

keywords = "(Kidney), Basement membrane, Fluorescence, Image processing, Immunohistochemistry",

author = "Basgen, {John M.} and Nevins, {Thomas E} and Michael, {Alfred F.}",

note = "Funding Information: We thank Christine Alonzi, Tom Groppoli, Sue Kupcho-Sandburg and Karen Clugge for excellent technical assistance, Paul Walker for the preparation and quantitation of radiolabeled IgG. We are also grateful to the staff of the Biomedical Image Processing Laboratory for help and assistance with the image analysis system and Dr. Ruth 0sterby for critical reading of the manuscript. This work was supported by grants from the National Institutes of Health (Al10704) and the Viking Children's Fund.",

year = "1989",

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doi = "10.1016/0022-1759(89)90188-9",

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T1 - Quantitation of antigen in tissue by immunofluorescence image analysis

AU - Basgen, John M.

AU - Nevins, Thomas E

AU - Michael, Alfred F.

N1 - Funding Information: We thank Christine Alonzi, Tom Groppoli, Sue Kupcho-Sandburg and Karen Clugge for excellent technical assistance, Paul Walker for the preparation and quantitation of radiolabeled IgG. We are also grateful to the staff of the Biomedical Image Processing Laboratory for help and assistance with the image analysis system and Dr. Ruth 0sterby for critical reading of the manuscript. This work was supported by grants from the National Institutes of Health (Al10704) and the Viking Children's Fund.

PY - 1989/11/13

Y1 - 1989/11/13

N2 - A method is described to measure antigen(s) in tissue section using an image analysis system to quantitate immunofluorescence following staining with fluorescein isothiocyanate (FITC)-conjugated antibody. The antigen utilized was an 125I-labeled goat anti-glomerular basement membrane antibody (1409 cpm/μg) administered intravenously to each of 11 Sprague-Dawley rats in doses ranging from 1.09 to 34.72 mg IgG. 24 h later, both kidneys were obtained for quantitative immunofluorescence following staining of tissue sections with FITC-labeled rabbit anti-goat IgG and for determination of radioactivity which reflects the amount of goat IgG present in isolated glomeruli. A linear correlation (r = 0.97) was observed between the dose of administered goat 125I-IgG and the amount bound to isolated glomeruli over the entire dosage range. A highly significant correlation (r = 0.98) was also seen between the mean brightness per glomerulus as determined by quantitative immunofluorescence and the amount of 125I-IgG bound per glomerulus but only at values less than 500 pg of IgG per glomerulus. Above these levels no correlation was observed, suggesting the presence of hidden epitopes in the bound goat IgG or the lack of availability of the FITC-labeled rabbit antibody.

AB - A method is described to measure antigen(s) in tissue section using an image analysis system to quantitate immunofluorescence following staining with fluorescein isothiocyanate (FITC)-conjugated antibody. The antigen utilized was an 125I-labeled goat anti-glomerular basement membrane antibody (1409 cpm/μg) administered intravenously to each of 11 Sprague-Dawley rats in doses ranging from 1.09 to 34.72 mg IgG. 24 h later, both kidneys were obtained for quantitative immunofluorescence following staining of tissue sections with FITC-labeled rabbit anti-goat IgG and for determination of radioactivity which reflects the amount of goat IgG present in isolated glomeruli. A linear correlation (r = 0.97) was observed between the dose of administered goat 125I-IgG and the amount bound to isolated glomeruli over the entire dosage range. A highly significant correlation (r = 0.98) was also seen between the mean brightness per glomerulus as determined by quantitative immunofluorescence and the amount of 125I-IgG bound per glomerulus but only at values less than 500 pg of IgG per glomerulus. Above these levels no correlation was observed, suggesting the presence of hidden epitopes in the bound goat IgG or the lack of availability of the FITC-labeled rabbit antibody.

KW - (Kidney)

KW - Basement membrane

KW - Fluorescence

KW - Image processing

KW - Immunohistochemistry

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ER -

Quantitation of antigen in tissue by immunofluorescence image analysis

Abstract

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